Microorganisms play a key role in phosphate circulation in the biosphere [Schink B (2005) Biological cycling of phosphorus. Met Ions Biol Syst 43, 131-151]. Their ability to accumulate and utilize inorganic polyphosphates (polyP) and orthophosphate (Pi) as phosphate reserves is one of the mechanisms of adaptation to changing environmental conditions [Kulaev I S, Vagabov V M, Kulakovskaya T V (2004): The biochemistry of inorganic polyphosphates. Wiley, Chichester. 2nd Edn.]. In recent years, due to rapid industrialization, urbanization, and population growth, water eutrophication i.e. excessive algal growth and related negative consequences in the surface water system has resulted in negative environmental effects. Obligatory anaerobic treatment of domestic and agro-industrial wastewater releases large amounts of phosphorus and nitrogen into wastewater. These nutrients are directly responsible for eutrophication of rivers, lakes, and seas worldwide [Lau P S, Tam N F Y, Wong Y S (1997) Wastewater nutrients (N and P) removal by carrageenan and alginate immobilized Chlorella vulgaris. Environ. Technol. 18, 945-951; Tre' panier C, Parent S, Comeau Y, Bouvrette J (2002). Phosphorus budget as a water quality management tool for closed aquatic mesocosms. Water Res. 36, 1007-1017]. Thus, removal of phosphorus from domestic and industrial wastewaters is central in curbing the water pollution problem of eutrophication.
Biological phosphate removal processes have been attracting attention in recent years because of low investment and operational costs compared with chemical precipitation processes [Pijuan M, Guisasola A, Baeza J A, Carrera J, Casas C, Lafuente J (2005) Aerobic phosphate release linked to acetate uptake: Influence of PAO intracellular storage compounds. Biochemical Engineering Journal. 26 (2-3)184-190 Engineering Bioreaction Systems: A Spanish Perspective; Michael H. Gerardi (2006) Wastewater Bacteria ISBN: 978-0-471-20691-0]. Bacteria involved in “enhanced biological phosphate removal” (EBPR) from waste accumulate large amounts of polyP, while some archaea and bacteria accumulate Pi [Smirnov A, Suzina N, Chudinova N, Kulakovskaya T, Kulaev I (2005) Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum. FEMS Microbiol. Ecol. 52, 129-137; Ryazanova L P, Smirnov A V, Kulakovskaya T V, Kulaev I S, (2007) Decrease of phosphate concentration in the medium by Brevibacterium casei cells. Microbiology. 76, 752-758; Ryazanova L P, Suzina N E, Kulakovskaya T V, Kulaev I S (2009) Phosphate accumulation of Acetobacter xylinum. Arch. Microbiol. 191, 467-471]. For understanding the role of bacteria in the phosphate turnover in nature and technology, it is important to reveal new species capable of effective phosphate accumulation. Independent studies have shown what is presumably the identical wild type isolated from activated sludge to have vastly different Phosphate uptake capabilities under laboratory conditions.
One striking feature which differs in these bioassays is the use of different media to conduct the experiments. Although mixed liquor is derived from the natural environment of phosphate-accumulating microorganisms (PAOs), synthetic media of different formulations are popular for experimental study of PAOs, due to their controllable consistencies.
Phosphate accumulating microorganisms are routinely screened using the following media—
M9 Medium [Minimal Medium 9]:
                Composition: (L−1) Glucose, 4 g; NaCl, 0.5 g; NH4Cl, 1 g; CaCl2, 0.014 g; MgSO4, 0.25 g; Na2HPO4, 12.8 g; KH2PO4, 3 g.            Reference: Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.NM Medium [Nakamura et al., 1995]:            Composition: (L−1) Glucose, 0.5 g; Peptone, 0.5 g; Monosodium glutamate 0.5 g, Yeast extract, 0.5 g; KH2PO4, 0.44 g; (NH4)2SO4, 0.1 g; MgSO4.7H2O, 0.1 g. pH of the medium is adjusted to 7.0 with dilute NaOH solution. Reference: Nakamura K, Hiraishi A, Yoshimi Y, Kawaharasaki M, Masuda K, Kamagata Y (1995) Microlunatus phosphovorus gen. nov., sp. nov., a New Gram-Positive Polyphosphate-Accumulating Bacterium Isolated from Activated Sludge. International Journal of Systematic Bacteriology, 45 (1) 17-22.SWW Medium [Synthetic Waste Water Medium]:        Composition: (L−1 tap water) Acetate, 177 mg; Peptone, 55 mg; CaCl2, 14.4 mg; MgSO4.7H2O, 95.0 mg; (NH4)2SO4, 12.7 mg; and KH2PO4, 21.1 mg.            Reference: Zhang H, Sekiguchi Y, Hanada S, Hugenholtz P, Kim H, Kamagata Y, Nakamura K (2003) Gemmatimonas aurantiaca gen. nov., sp. nov., a Gram-negative, aerobic, polyphosphate-accumulating micro-organism, the first cultured representative of the new bacterial phylum Gemmatimonadetes phyl. nov. International Journal of Systematic and Evolutionary Microbiology, 53, 1155-1163.
Literature reports depict that various mixed liquor and synthetic media have been used in microbial Phosphate uptake research [Ohtake H, Takahashi K, Tsuzuki Y, Toda K (1985) Uptake and release of phosphate by a pure culture of Acinetobacter calcoaceticus. Water Res. 19 (12) 1587-1594; Appeldoorn K J, Kortsee G J J, Alexander J B Z (1992) Biological phosphate removal by activated sludge under defined conditions. Water Res. 26 (4) 453-460; Muyima N Y O, Cloete T E (1995) Phosphate uptake by immobilized Acinetobacter calcoaceticus in a full scale activated sludge plant. J. Ind. Microbiol. 15, 19-24; Morohoshi, T., Yamashita T, Kato J, Ikeda T, Takiguchi N, Ohtake H, Kuroda A (2003) A method for screening phosphate-accumulating mutants which remove phosphate efficiently from synthetic wastewater. J. Biosci. Bioeng., 95(6), 637-640]. However, no conclusive studies have been conducted to compare the effect of these media types on bacterial response, or if a particular medium causes results to be inflated or exaggerated. The use of synthetic media and mixed liquor in different studies resulted in conflicting reports on the Phosphate uptake capabilities of PAOs isolated from the environment.
Thus, from the findings of the prior art it may be summarized that there is a paucity of a medium and method for screening phosphate-accumulating microorganisms (PAOs) from the environment. Therefore, a new screening strategy is needed towards isolating effective PAOs in environments with diverse microbial populations and limited organic resources. It is noteworthy to mention here that PAOs are often out-competed by organisms capable of utilising available nutrients more rapidly. Further, screening microbial isolates for phosphate accumulation by quantitative methods requires investment of time, labour and chemicals. In view of these problems, the inventors of the present invention realized that there exists a dire need to develop a medium optimal for efficient screening of P-accumulating microorganisms, which is rapid and allows many PAOs to be screened, based upon visual observations.